The disclosures of publications listed at the end of this specification immediately preceding the claims are hereby incorporated by reference in their entireties into this specification in order to more fully describe the state of the art to which this invention pertains.
Insulin is a polypeptide hormone essential for the control of glucose metabolism and it is administered daily to patients suffering from diabetes mellitus, a metabolic disorder characterized by an inadequate supply of insulin.
In vivo, the hormone is first synthesized as a long precursor molecule, subsequently processed to its biologically active form, consisting of an A and a B chain. In more detail, the gene for preproinsulin is transcribed in the beta cells of the endocrine pancreas into an mRNA precursor, which is then spliced to produce mature mRNA. This mRNA is translated into preproinsulin (NH.sub.2 -preregion-B chain-C peptide-A chain-COOH), which is sequentially processed into proinsulin and finally into insulin. The first step in the processing is the proteolytic elimination of the preregion, which serves as a hydrophobic signal sequence for the transfer of the nascent chain through the microsomal membranes of the rough endoplasmatic reticulum. In human preproinsulin, the length of the preregion is 24 amino acids.
In proinsulin, the two regions of the polypeptide chain that will become the mature insulin, the B- and A chains, are connected to each other by the C peptide (or C-chain), which comprises at the N and C termini two pairs of basic amino acids. In most C-peptides, these pairs are Arg-Arg and Lys-Arg. The human C peptide, including the two flanking pairs of basic amino acids, contains 35 amino acids. The C peptide connects the two portions of the polypeptide in order to aid in appropriate disulfide bridge formation between the B and A segments. Therefore the role of the C peptide does not depend greatly on its structure. In fact, its replacement by a shorter synthetic bridge still allows proper folding of the proinsulin molecule (1,2).
The proinsulin folds with the concomitant oxidation of two interchain disulfide bonds and of one disulfide bond within the A chain. In the last stage of maturation, proteolytic enzymes cleave at the basic amino acids to release the C peptide and form the mature insulin (3). In human insulin, the A chain is 21 amino acids long while the B chain is 30 amino acids long.
World demand for insulin exceeds several tons annually and there is a severe shortage of supply. Traditionally, insulin was produced from limited animal sources, mainly bovine and porcine pancreatic preparations, which differ from human insulin and may elicit an adverse immune reaction.
Studies carried out during the 1960's demonstrated in vitro production of insulin. Insulin synthesis was achieved by combining the A and B chains in their S-sulfonated forms (4) or by the spontaneous reoxidation of reduced proinsulin (5). The latter method was not practical for large scale insulin production due to very low protein concentration in the oxidation mixture. Insulin could subsequently be recovered following treatment with trypsin and carboxypeptidase B (6).
Semi-synthetic and biosynthetic (recombinant) human insulin have recently become available. Semi-synthetic human insulin is produced from porcine insulin by the trypsin catalyzed exchange of alanine with threonine at position 30 of the B chain (the only difference between porcine and human insulin). The recombinant human insulin produced either in E. coli or yeast will eventually replace all other routes of manufacture.
Biosynthetic recombinant human insulin is currently manufactured by two routes: either by producing the A and B chains separately in E.coli and subsequently combining them (7,8), or by enzymatic conversion of pro-insulin like polypeptides expressed in either E.coli (1,8) or yeast (2,9).
In most cases proinsulin is produced as a hybrid protein which accumulates as intracellular precipitated protein. This hybrid is normally purified and cleaved by CNBr in order to release the proinsulin polypeptide. The latter is further modified by oxidative sulfitolysis to proinsulin S-sulfonate. The proinsulin S-sulfonate is then purified and folded, under reducing conditions, to proinsulin (8). Conversion of the proinsulin to insulin is achieved by the combined action of trypsin and carboxypeptidase B (6).
Patent Publication No. EP 195691 B1, assigned to Novo Nordisk A/S describes a proinsulin of the formula B-Lys-Arg-A and the use thereof for the preparation of insulin in yeast.
Patent Publication No. EP 196056 Bi, assigned to Chiron Corp., describes an hSOD-proinsulin protein produced by yeast. The hSOD-proinsulin protein is subjected to cyanogen bromide cleavage and sulfitolysis prior to folding. Hoechst discloses in EPO Publication No. 379162 that `false recombinants of insulin precursors` (i.e. recombinant insulin products with incorrect or partially incorrect intermolecular disulfide bridges) can be converted to `correct` insulin products without sulfitolysis by reacting the false recombinants with excess mercaptan in an aqueous medium in the presence of an organic redox system. The original sulfitolysis step takes place after the amino acid or peptide radical is cleaved off (chemically or enzymatically) from the fusion polypeptide (which takes place after lysis of the host cell) since then the six cysteines of the insulin precursor are converted into their S-sulfonates. In a subsequent renaturing step, natural proinsulin is produced from this proinsulin S-sulfonate by formation of the three correct disulfide bridges. During this renaturing step, the so-called `false recombinants` are produced.
Hoechst further discloses, in PCT International Publication No. WO 91/03550, a process for the preparation of fusion proteins containing a desired protein (e.g. proinsulin) and a "ballast constituent". Sulfitolysis is carried out before folding while the "ballast constituent" is cleaved off concomitantly with the C-chain of the proinsulin, after folding.
In addition, Hoechst describes in EP 347781 B1, a "mini-proinsulin" (B-Arg-A) and the use thereof for the preparation of mono-Arg insulin and insulin. They further describe fusion proteins which comprise B-Arg-A and a "ballast constituent". The "ballast constituent" is cleaved off by cyanogen bromide and sulfitolysis is carried out before folding of the polypeptide.
The subject invention discloses recombinant human insulin production by an improved and efficient process. Recombinant proinsulin hybrid polypeptides comprising a leader sequence are synthesized in E.coli. After partial purification, they are folded with the leader peptide still attached under conditions which permit correct folding. Biologically active human insulin is then produced by combined treatment with trypsin and carboxypeptidase B in which these enzymes cleave off the leader peptide and the C-chain concomitantly. The purified human insulin thus produced is identical to naturally occurring human insulin.
The hazardous and cumbersome procedures involved in CNBr cleavage of hybrid polypeptides and sulfitolysis used to protect the abundant SH groups are excluded from this novel process since the entire proinsulin hybrid polypeptide can fold efficiently into its native structure even in the presence of the leader peptide and the unprotected cysteine residues. The active recombinant human insulin is released by enzymatic cleavage and is thereafter purified.